Project Summary/Abstract The main purpose of this proposal is to gain further insight into the unique epibiotic parasitic interaction between the recent, ?yet to be cultivated? bacterial pair, XH001, an Actinomyces odontolyticus subsp. actinosynbacter strain, and TM7x, a candidate TM7 phyla member. The accomplishment of the NIH Human Microbiome Project has demonstrated the staggering amount of the microbial diversity on planet earth and in the human body. Since there are only a minute number of microbial species that can be cultured and studied in vitro, there is an immense gap in our understanding between available genomic information versus recognized physiological and pathological functions. TM7x as well as other related Actinomyces species have been found in various environmental and human body sites as well as associated with a variety of mucosal diseases such as periodontitis, vaginosis, and inflammatory bowel disease. This proposed study will dramatically increase our understanding of novel, ?yet to be cultivated? bacteria and define TM7x?s pathogenicity in the human host. Our lab recently co-cultivated the first known TM7 representative along with XH001 from the oral cavity. The inability to cultivate pure TM7x separate from XH001, suggests that XH001 is an obligate symbiotic host for TM7x. To date this relationship has never been observed before and merits further investigation. Moreover, XH001, like other Actinomyces species, induces TNF-? expression in macrophages; however, in the presence of TM7x, this TNF-? expression is diminished, suggesting TM7x may possess immunomodulatory capabilities. A preliminary transcriptomic study, comparing XH001 monoculture vs. XH001 associated with TM7x, revealed the most highly upregulated gene, an lsrB homologue which encodes a periplasmic binding protein to recognize auto-inducer 2 (AI-2) molecules, indicating that AI-2 quorum sensing maybe directly involved in their association. Preliminary data indicates that not only is the XH001lsrB mutant producing low levels of the AI-2 signal, but when associated with TM7x in co-culture, it forms a statistically significant deficient biofilm compared to wild type XH001 co-cultured with TM7x. Therefore, we hypothesize that the AI-2 signaling system plays a crucial role in the establishment and maintenance of the epibiotic-parasitic lifestyle with its host species XH001. The three following interconnected aims will be used to address this hypothesis: (1) Understanding the genetic regulation of AI-2 signaling during the multi-stage XH001 growth phases with TM7x using XH001lsrB and XH001luxS mutants. (2) Elucidate other genes involved in biofilm formation, production of virulence factors, and maintenance/establishment of the epibiotic parasitic relationship of TM7x/XH001 by conducting global gene expression analysis using AI-2 deficient genetic mutants. (3) Use in vitro cell culture models to study pathogenesis-related phenotypes, including the ability to induce the production of pro-inflammatory cytokines (MCP-1, IL-8, and TNF-?), and adherence to and invasion of selected host cells lines.